We have discovered a new series of analogues of the alkaloid methyllycaconitine (MLA) that act as antagonists at the neuronal nicotinic acetylcholine receptor (nAChR). Our goals for this proposal are to determine the structural aspects of MLA that confer nicotinic antagonist activity. Our hypothesis is that MLA analogues incorporating more of the MLA structure ("more MLA-like") will be more potent and selective antagonists at the alpha7 nAChR. Our long-term goals are to understand the structural determinants of small molecule - nicotinic receptor binding and use this understanding to design and synthesize novel pharmaceutical agents. We propose to prepare novel analogues based upon the structure of MLA. These compounds will include ring E analogues as well as analogues incorporating rings C, B, D, and F. These new analogues should have excellent potency at the apha7 nAChR. We propose to prepare ring E analogues of MLA with improved potency at the alpha3 nAChR. We will evaluate the potency and receptor subtype specificity of MLA analogues. Analog activity and subtype specificity will be assessed through both binding and functional studies using model systems expressing both CNS and peripheral nAChRs. Overall, we have found a simple analogue of MLA that should be useful in assessing structural requirements necessary for potency and nAChR subtype selectivity. We have prepared four series of MLA analogues and assayed these compounds for both potency and selectivity at the alpha7, alpha3, and alpha4 nAChR. These preliminary studies further define the structural importance of substitution on the nitrogen of the piperidine ring and substitution on the succinimide ring. We have developed a binding assay for the alpha3 nAChR which should be useful for our work as well as the general study of nicotinic ligand selectivity. With the support of our R03 grant we have met our initial goals, demonstrating that we can both prepare and evaluate analogues of MLA as nicotinic antagonists. Further we have shown that several of these compounds are among the most potent nicotinic antagonists known. We have also shown that the potency of these compounds can be improved through simple modifications.